The long-term goals of this research are to understand how Staphylococcus aureus and its superantigens (SAgs), cytotoxins, and other exoproteins interact with mucosal surfaces to facilitate SAg penetration to cause serious human illnesses. We propose to use the vaginal epithelium as the model multi-layered mucosal surface, and hypothesize the data obtained in planned studies will also be applicable to other mucosal surfaces. Prior results using an ex vivo porcine vaginal permeability model indicate that the SAg, TSST-1, traverses mucosal surfaces in small amounts, but localizes in higher amounts within the inner epithelium, possibly serving as a toxin reservoir. In the presence of staphylococcal 1-toxin, TSST-1 penetrated the full thickness of the vaginal mucosa in significantly higher amounts, with considerable TSST- 1 also remaining within the epithelium. We hypothesize that cytotoxic and inflammatory properties of staphylococcal cytotoxins and other exoproteins enhance penetration of TSST-1 to inner epithelial cells where TSST-1 binds to an undescribed epithelial cell receptor, triggering pro-inflammatory cytokine production. Base on these hypotheses we propose two specific aims: 1) To determine the contribution of S. aureus cytotoxins (exoproteins) in enhancing the penetration of TSST-1 across epithelial surfaces by causing cytolytic and/or inflammatory effects on epithelial cells, and 2) To determine TSST-1 receptor binding and the mechanism of TSST-1 stimulation of epithelial cells, leading to intracellular activation of signaling pathways with subsequent cytokine production. Our preliminary data suggest that the most likely candidate exoproteins to facilitate penetration of TSST-1 across mucosal barriers are cytotoxins, including 3-toxin, which we propose to study in depth through use of purified 3-toxin components and allelic replacement knockout strains. In the course of these studies, we will also study mucosal penetration and activities of a deletion [missing amino acids 1 to 72] mutant TSST-1 protein (13,000 MW vs. 22,000 MW wild-type TSST-1) that maintains superantigenicity and is associated with a rapidly progressive, 100% fatal, extreme pyrexia syndrome in humans. We believe the planned studies will clarify the mechanisms by which TSST-1 penetrates mucosal barriers to cause large numbers of human diseases. These studies may suggest novel therapeutic strategies to manage serious S. aureus illnesses. PUBLIC HEALTH RELEVANCE: The bacterium Staphylococcus aureus often initiates serious human diseases from the organism's colonization of mucous membranes, followed by secretion of potent exotoxins referred to as superantigens. Illnesses include toxic shock syndrome that may have both menstrual and non- menstrual forms, and that may affect thousands of humans each year in the United States. This application studies the mechanism by which superantigens penetrate mucosal surfaces, allowing S. aureus to cause life threatening toxic shock syndrome, and possibly suggesting novel strategies to prevent and manage such illnesses.